Aqueous compositions that maintains fidelity of the nucleic acid sequences of a biological specimen

ABSTRACT

Disclosed are compositions for isolating populations of nucleic acids from biological, forensic, and environmental samples. Also disclosed are methods for using these compositions as one-step formulations for killing pathogens, inactivating nucleases, and releasing polynucleotides from other cellular components within the sample, and stabilizing the nucleic acids prior to further processing or assay. The disclosed compositions safely facilitate rapid sample collection, and provide extended storage and transport of the samples at ambient or elevated temperature without contamination of the sample or degradation of the nucleic acids contained therein. This process particularly facilitates the collection of specimens from remote locations, and under conditions previously considered hostile for preserving the integrity of nucleic acids released from lysed biological samples without the need of refrigeration or freezing prior to molecular analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/969,339 filed Dec. 15, 2015, which issued as U.S. Pat. No. 9,683,256Jun. 20, 2017, which is a continuation-in-part of U.S. application Ser.No. 13/328,992 filed Dec. 16, 2011, which issued as U.S. Pat. No.9,416,416 Aug. 16, 2016, which is a continuation of U.S. applicationSer. No. 12/426,890 filed Apr. 20, 2009, which issued as U.S. Pat. No.8,080,645 Dec. 20, 2011, which is a continuation-in-part of U.S.application Ser. No. 12/243,949, filed Oct. 1, 2008, which issued asU.S. Pat. No. 8,084,443 on Dec. 27, 2011, which claims the benefit ofU.S. Provisional Application No. 60/976,728, filed Oct. 1, 2007, and theentire contents of each application is specifically incorporated hereinin its entirety.

FIELD OF THE INVENTION

The invention relates to aqueous compositions for collection, transport,and storage of a biological specimen containing a population of nucleicacids in a single reaction vessel, which can then be purified and/oranalyzed using conventional molecular biology methods. In particular,the invention is directed to a one-step composition that a) inactivatesviruses or microbes in the sample, b) lyses the biological cells ortissues to free the nucleic acids from cellular debris and extraneousbiomolecules, c) protects the nucleic acids from degradation byendonuclease activity, and d) preserves the nucleic acids for subsequentisolation, detection, amplification, and/or molecular analysis. In aparticularly advantageous application, all four functions may beachieved in a single composition, and in a single reaction vessel, andthe resultant sample may be stored at ambient temperature for extendedperiods without significant degradation of the polynucleotides containedwithin the sample.

BACKGROUND OF THE INVENTION

In the field of molecular and diagnostic analysis, the ability to keepnucleic acids in a biological sample stable, whether the specimen istaken in a remote field location, a doctor's office or in a laboratory,often determines whether the nucleic acids can be successfully analyzed.Nucleic acids in a biological sample quickly degrade and/or denature atroom temperature and must generally be stored under freezingtemperatures to remain stable; however, some degree of degradation stilloccurs over time. This problem is magnified when a specimen is collectedat a remote field site, or a significant distance from a doctor's officeor laboratory environment, and especially where there may be limited, orno, access to consistent and constant cooler/refrigerator/freezerconditions until the sample is analyzed, such as where access to power(i.e., electricity), or freezer equipment is not constant or isnon-existent. The problem is yet further magnified when the desirednucleic acids for downstream analysis include ribonucleic acid (RNA),which is particularly susceptible to degradation, e.g., by endogenous orexogenous endonuclease activity. Specimen transport technology presentlyavailable in the art often uses special transport media for biologicalsamples for transport to a laboratory, in particular, packaging thatimposes short time, low temperature, and practicality limits.

In addition to concerns regarding specimen stability, often there areadditional concerns regarding the reagents that are used to store and/ortransport the collected samples. For example, the reagents themselvesfrequently require cold temperatures or other special care to maintainstability. Due to these stability issues, for example, transport of thereagents to a field site, storage at the field site before use, andtransport of the biological specimens and reagents back to a testingsite is a primary concern.

Another significant concern when working with biological specimens isthe potential inoculation, release, or dissemination of live infectiouspathogens or biological agents from the specimen into the environment.Specific protocols currently exist that are employed when handlingsamples that may be infectious or otherwise pose health or safety risks.If the sample is kept viable and/or biologically intact to preserve itsintegrity for testing, individuals involved in the collection, transfer,and testing process are potentially exposed to highly dangerouscontagions. Additionally, innocent bystanders nearby a field site (ornearby during transport) can be exposed if a release of the contagionoccurs. As a result, the required safety measures typically increase theexpense and effort required to move such samples from one location toanother.

Until recently, clinical laboratory methods for pathogen detection werelabor-intensive, expensive processes that required highly knowledgeableand expert scientists with specific experience. The majority of clinicaldiagnostic laboratories employed the use of traditional culturingmethods that typically require 3 to 7 days for a viral culture—and evenlonger for some other bacterial targets. Furthermore, traditionalculturing requires collection, transport, and laboratory propagation andhandling of potentially infectious biological agents such as Ebola,avian influenza, severe acute respiratory syndrome (SARS), etc.

The field of clinical molecular diagnostics changed drastically with theadvent of polymerase chain reaction (PCR) in the mid eighties, however,and shortly thereafter with real-time PCR in the mid 90's. Real-time PCR(and RT-PCR) can deliver results in hours, and the majority of moderndiagnostic laboratories are transitioning away from traditional culture,and into nucleic-acid-based detection platforms, such as real-time PCR.Recent improvements in detection chemistries, such as new and improvedreporting/quenching fluors, minor groove binders (MGB), and stabilizedamplification reagents have paved the way for more sensitive andspecific pathogen detection assays that have been proven more timely,robust, and economical than antiquated culturing methods. Advances inother nucleic acid detection strategies (in addition to real-time PCR)such as transcription-mediated amplification, ligase chain reaction(LCR), microarrays, and pathogen gene chips, have also contributed to atransition from culture vials in the clinical laboratory.

Several commercial companies (e.g., Qiagen, Roche, and bioMérieux) havedeveloped automated nucleic acid extraction instruments, and haveattempted to automate the parts of the multi-part process from sampleisolation to molecular analysis. For example, the Tigris DTS®(Gen-Probe, San Diego, Calif., USA) automates the entire detectionprocess, and in late 2004 was FDA approved for use with Gen-Probe'sAPTIMA COMBO 2® assay, an FDA-approved amplified nucleic acid test (NAT)for simultaneously detecting Chlamydia trachomatis and Neisseriagonorrhoeae.

Accordingly, there is a need in the art for a safe collection, storageand transport system that maintains the integrity of the nucleic acidsof even a dangerous biological specimen, typically for further molecularanalysis or diagnostic testing, without the need for freezing thecollected biological specimen, the collection reagents, or the collectedsample in the reagents, without posing a risk to workers or innocentbystanders, and allowing for the use of less expensive and moreconvenient transportation methods or complicated shipping precautions.

SUMMARY OF THE INVENTION

The present invention encompasses new and useful compositions, as wellas methods of making and employing them, that may advantageously improveconventional collection, lysis, transport and storage methods for thepreparation of nucleic acids from one or more biological sources.Accordingly, the present invention advantageously can provide acollection and preservation formulation to inactivate and lyse abiological specimen containing nucleic acids, and preserve nucleic acids(RNA/DNA) within the biological specimen, preferably all in a singlereaction vessel, such that the integrity of the nucleic acids is atleast substantially maintained, and preferably entirely maintained, sothat a portion of the nucleic acids are readily available for moleculardiagnostic analysis.

An additional advantage of the present invention is that the formulationcan enable the separated or released nucleic acids to remain at leastsubstantially stable, without requiring consistent and constant coolertemperatures, such as refrigeration or freezing.

The one-step formulations disclosed herein accomplish the following mainfunctions: inactivation or killing of pathogens within the sample; lysisof cells and separation or release of nucleic acids from the cells;inactivation of endogenous or exogenous nucleases and other cellularenzymes to prevent degradation of the nucleic acids present in thesample; and facilitation of collection and handling of the sample atambient temperatures, stabilization of the nucleic acids duringsubsequent transport and storage of the sample, andpreservation/maintenance of the integrity of one or more polynucleotidescontained with the liberated nucleic acids.

The ability to achieve all of these desirable functions in a single-stepformulation, preferably in a single reaction zone or reaction vessel, isa particularly marked advantage over that presently available. Presentlyexisting technologies do not include a single-step composition thatprovides for inactivation of biological components containing nucleicacids, release of nucleic acids through lysis of cells and separation orrelease of nucleic acids, and maintenance of the integrity of thenucleic acids. Without being bound by theory, this is in part believedto be because the process of killing the biological organism present ina sample typically results in release and activation of enzymes thatdegrade proteins and nucleic acids. Enzymatic degradation leads tosample destruction, which prevents analysis. The present invention,however, stabilizes and preserves the integrity of nucleic acids presentin the specimen for diagnostic testing.

The one-step formulations of the present invention allow for preferablysimultaneous inactivation of biological components containing nucleicacids, lysis and separation or release of nucleic acids, stabilization,and preservation. In one embodiment, some or all of the inactivation,lysis and separation or release, stabilization, and preservation, aresequential. In a preferred embodiment, however, a majority or preferablyall of these functions occur simultaneously. In all embodiments, theone-step formulation is combined with the sample to initiate thesefunctions. This is in contrast to previous technology in whichinactivation did not necessarily occur, and lysis, stabilization, andpreservation occurred in a succession of separate steps, each steptypically using one or more distinct reagents and protocols that wereseparately added.

The sequential format of prior procedures was needed to minimize errors,avoid reagent incompatibility, and provide stepwise control of results.The present invention provides all these benefits and adds the furtherbenefits of maintaining the integrity of the nucleic acids, renderingthem ready for extraction and purification, thereby improving theirultimate yield. The one-step formulation's preferably simultaneousinactivation of biological components containing nucleic acids, lysisand release of nucleic acids from cellular debris, stabilization, andpreservation of nucleic acids reduces the chance for degradation of theRNA/DNA in the sample that may occur during lysis, or after lysis andbefore stabilization, which contributes to improved yield of the nucleicacids that are eventually extracted. An improved yield can lead tosuperior test results.

In one embodiment, the invention provides a composition that includes:a) one or more chaotropes (each preferably present in the composition anamount from about 0.5 M to about 6 M); b) one or more detergents (eachpreferably present in the composition an amount from about 0.1% to about1%); c) one or more chelators (each preferably present in thecomposition in an amount from about 0.01 mM to about 1 mM); d) one ormore reducing agents (each preferably present in the composition in anamount from about 0.05 M to about 0.3 M); and e) one or more defoamingagents (each preferably present in the composition in an amount fromabout 0.0001% to about 0.3%).

Exemplary chaotropes include, without limitation, guanidine thiocyanate(GuSCN), guanidine hydrochloride (GuHCl), guanidine isothionate,potassium thiocyanate (KSCN), sodium iodide, sodium perchlorate, urea,or any combination thereof. Descriptions of additional exemplarychaotropes and chaotropic salts can be found in, inter alia, U.S. Pat.No. 5,234,809 (specifically incorporated herein in its entirety byexpress reference thereto).

Exemplary detergents include, without limitation, sodium dodecyl sulfate(SDS), lithium dodecyl sulfate (LDS), sodium taurodeoxycholate (NaTDC),sodium taurocholate (NaTC), sodium glycocholate (NaGC), sodiumdeoxycholate (NaDC), sodium cholate, sodium alkylbenzene sulfonate(NaABS), N-lauroyl sarcosine (NLS), salts of carboxylic acids (i.e.,soaps), salts of sulfonic acids, salts of sulfuric acid, phosphoric andpolyphosphoric acid esters, alkylphosphates, monoalkyl phosphate (MAP),and salts of perfluorocarboxylic acids, anionic detergents includingthose described in U.S. Pat. No. 5,691,299 (specifically incorporatedherein in its entirety by express reference thereto), or any combinationthereof.

Exemplary reducing agents include, without limitation, 2-mercaptoethanol(β-ME), tris(2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT),formamide, dimethylsulfoxide (DMSO), or any combination thereof. In apreferred embodiment, the reducing agent includes or is TCEP.

Exemplary chelators include, without limitation, ethylene glycoltetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid(HEDTA), diethylene triamine pentaacetic acid (DTPA),N,N-bis(carboxymethyl)glycine (NTA), ethylenediaminetetraacetic (EDTA),citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate,ammonium bicitrate, citric acid, diammonium citrate, potassium citrate,magnesium citrate, ferric ammonium citrate, lithium citrate, or anycombination thereof. In preferred embodiments, the chelator includesEDTA, a citrate, or a combination thereof. In a more preferredembodiment, the chelator includes EDT.

The compositions of the invention can further include a defoaming agentto prevent the formation of bubbles that typically result from thepresence of detergents in the formulation. Defoaming agents facilitatepipetting and handling of the disclosed compositions. Exemplarysurfactants/defoaming agents include, without limitation,cocoamidopropyl hydroxysultaine, alkylaminopropionic acids, imidazolinecarboxylates, betaines, sulfobetaines, sultaines, alkylphenolethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropyleneglycols, polyoxyethylenated mercaptans, long-chain carboxylic acidesters, alkonolamides, tertiary acetylenic glycols, polyoxyethylenatedsilicones, N-alkylpyrrolidones, alkylpolyglycosidases, silicone polymerssuch as Antifoam A®, or polysorbates such as Tween®, or any combinationthereof. In a preferred embodiment, a defoaming agent includes asilicone polymer.

Optionally, the compositions of the invention may further include one ormore buffers (each preferably present in the final composition in anamount from about 1 mM to about 1 M). Exemplary buffers include, withoutlimitation, tris(hydroxymethyl) aminomethane (Tris), citrate,2-(N-morpholino)ethanesulfonic acid (MES),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic Acid (BES),1,3-bis(tris(hydroxymethyl)methylamino)propane (Bis-Tris),4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine),N-2-acetamido-2-iminodiacetic acid (ADA),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES),piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), bicarbonate,phosphate, or any combination thereof. In a preferred embodiment, thebuffer includes a citrate.

The inclusion of one or more of such optional but preferred buffers isdesirable to control the pH of the formulations, since it has been foundthat nucleic acid extraction is optimal in a pH range of about 5 to 7.Preferably, the one or more buffers employed in the disclosedcompositions are chosen to provide a significant buffering capacity inthe range from a pH of about 6 to a pH of about 8, more preferablywithin a pH range of about 6 to about 7, and more preferably still,within a pH range of about 6.2 to about 6.8. In exemplary embodiments,the pH of PrimeStore™ Solutions (also referred to herein as “PSS”) ispreferably about 6.7±0.25.

The compositions of the invention may also further optionally includeone or more short-chain (preferably from 1- to 6-carbon [i.e., C₁-C₆]alcohols) alkanols (each preferably present in the composition in anamount from about 1% to about 25%, although higher percentages of thealcohols may be employed if desired). Exemplary short-chain alkanolsinclude linear and branched-chain alcohols, such as, without limitation,methanol, ethanol, propanol, butanol, pentanol, hexanol, or anycombination thereof.

The compositions of the invention may also further optionally includeone or more additional compounds or reagents including, withoutlimitation, betaine, bovine serum albumin, and osmolytes such astrehalose, sorbitol, and the like.

In certain embodiments, the addition of nucleic acids (e.g., RNA and/orDNA) is contemplated to be beneficial for a variety of purposes andapplications of the disclosed methods: a) as a “carrier” (The additionof small amounts of supplemental RNA/DNA has been previously been shownto augment/increase the overall yield of samples/specimens, particularlyoriginal specimens that may contain low amounts of target, i.e., cells,viruses, bacteria); b) as an internal positive control for downstreammolecular processes and to track or monitor the fidelity of the nucleicacid preparation from sample collection to detection; and c) forcomparison to a ‘calibrator’ for downstream quantitative analysis, e.g.,qRT-PCR and the like. In such embodiments, one or more known or“control” nucleic acids could be added to the compositions in a finalconcentration of from about 1 pg to about 1 μg.

Preferably, the compositions of the invention provide sufficientbuffering capacity to adequately stabilize the populations ofpolynucleotides obtained from a sample, and will, most preferably, bebuffered to a pH of about 6.4 to 6.9 during formulation, and willmaintain the isolated populations of polynucleotides in a similar pHrange when the sample is contacted with the storage/collectionformulations described herein.

Preferably, the collected samples will include one or more populationsof nucleic acids that are isolated from a biological sample, specimen,or source, including, for example, RNAs and DNAs.

The compositions of the present invention will typically at leastsubstantially inactivate, and preferably entirely inactivate, anyendogenous or exogenous RNAses or DNAses present in the sample, suchthat the nucleic acids of the sample are substantially free of anydegradation, and preferably do not degrade, or lose integrity, duringthe collection, lysis, storage, and transport of the sample forsubsequent in vitro or in vivo analyses.

Exemplary formulations of the invention include a one-step collectionsolution that lyses, stabilizes, and preserves the integrity of nucleicacids prepared from a biological sample for subsequent RNA and/or DNAanalysis.

The disclosed compositions were developed and optimized, inter alia: 1)to facilitate preparation of high-quality nucleic acids from clinical orenvironmental specimens, 2) to inactivate, kill, or otherwise neutralizepotentially infectious pathogens in a biological sample to facilitatesafe handling and transport of the collected specimens, and 3) tostabilize released (i.e., ‘naked’) DNA/RNA for prolonged periods withouthydrolysis or nuclease degradation of the released nucleic acids.

The compositions described herein are ideal for clinical, field anddeployment use, or for high volume sample collection/extraction.Specimens collected in one or more of the disclosed compositions arebiologically inactivated, and may be safely shipped, typically evenwithout refrigeration or dry ice.

Exemplary formulations of the storage/transport/collection compositionsof the invention are described in the examples herein, and include,without limitation, a composition that includes about 4 M of a chaotrope(such as guanidine thiocyanate, guanidine hydrochloride, guanidineisocyanate, or any combination thereof), about 10 mM to 30 mM of achelator (such as EGTA, HEDTA, DTPA, NTA, EDTA, citrate anhydrous,sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate,citric acid, diammonium citrate, ferric ammonium citrate, lithiumcitrate, or any combination thereof), about 0.25% of a detergent (suchas SDS, LDS, NaTDC, NaTC, NaGC, NaDC, sodium cholate, NaABS, NLS, or anysalt or combination thereof), about 0.1 M of a reducing agent (such asβ-ME, DTT, DMSO, formamide, TCEP, or any combination thereof), and about0.1% of a surfactant/defoaming agent (such as a silicone polymer [e.g.,Antifoam A®] or a polysorbate [e.g., Tween®], or any combinationthereof).

Additional exemplary formulations of the specimen collectioncompositions of the invention include, without limitation, a compositionthat includes about 3 M of a chaotrope (such as guanidine thiocyanate,guanidine hydrochloride, guanidine isocyanate, or any combinationthereof), about 1 mM of a reducing agent (such as β-ME, TCEP, formamide,DTT, DMSO, or any combination thereof), about 1 to 10 mM of a chelator(such as EGTA, HEDTA, DTPA, NTA, EDTA, citrate anhydrous, sodiumcitrate, calcium citrate, ammonium citrate, ammonium bicitrate, citricacid, diammonium citrate, ferric ammonium citrate, lithium citrate, orany combination thereof), about 0.25% of a detergent (such as SDS, LDS,NaTDC, NaTC, NaGC, NaDC, sodium cholate, NaABS, NLS, or any salt orcombination thereof), and optionally but preferably about 0.0002% of adefoaming agent (also referred to as an antifoaming agent) (such as asilicone polymer or a polysorbate, or any combination thereof) and about100 mM of a buffer (such as Tris, MES, BES, Bis-Tris, HEPES, MOPS,bicarbonate, citrate, phosphate, or any combination thereof).

Another exemplary formulation of the disclosed polynucleotide isolationand stabilization compositions include, without limitation, acomposition that includes about 1 to about 4 M of a chaotropic agentsuch as guanidine thiocyanate, guanidine hydrochloride, or guanidineisocyanate; about 0.5 to 100 mM of a chelating agent such as EDTA, orsodium citrate, or both; about 0.1 to about 1% of an anionic detergentsuch as SDS or N-lauroyl sarcosine, sodium salt; about 0.001 to about0.0001% of a surfactant or wetting agent such as the silicone polymer,Antifoam A®, e); about 10 to about 500 mM of a buffering agent such asTris-HCl; and about 10 to about 25% of a short-chain alkanol such asethanol.

In particular embodiments, the invention provides a composition thatincludes about 3 M guanidine thiocyanate; about 1 mM TCEP; about 10 mMsodium citrate; about 0.5% N-lauroyl sarcosine, sodium salt; about0.0002% Antifoam A, about 100 mM Tris-HCl, about 0.1 mM EDTA; and about23% ethanol.

The invention also provides a method for obtaining a population ofpolynucleotides from a sample suspected of containing nucleic acids. Themethod generally involves associating the sample with an amount of oneof the disclosed compositions, under conditions effective to obtain apopulation of polynucleotides from the sample. The invention does notrequire separation of the population to “obtain” the sample, as laterdiagnosis may or may not need such separation.

The invention also provides a method of preparing a one-step aqueousformulation of the collection/lysis/transport/storage compositionsdescribed herein for the collection of nucleic acids such as RNA and/orDNA. In an overall sense, the method generally involves combining one ormore chaotropes and nuclease-free water at a temperature of about 20° C.to 90° C. in a reaction zone; then combining the dissolved one or morechaotropes with one or more reducing agents, one or more chelators, andone or more detergents in the reaction zone to form an intermediatecomposition; optionally combining a silicone polymer with theintermediate composition in an amount sufficient to minimize foamingduring further preparation of the one-step aqueous formulation;combining a sufficient amount of buffer to the intermediate compositionto maintain a pH of about 6 to 6.9; optionally combining a secondchelating agent to the reaction zone; then increasing the temperature ofthe second intermediate composition to about 60 to 95° C. for about 1 to30 minutes and lowering the temperature to ambient conditions;optionally then combining a C₁₋₆alcohol with the contents of thereaction zone; and optionally adjusting the pH to be about 6.4 to 6.9.

In additional embodiments, the invention provides a method for preparingone-step aqueous formulations adapted to obtain a population ofpolynucleotides from a biological sample that is suspected of containingnucleic acids. This method generally involves at least the steps of: a)contacting the sample with an amount of the one-step aqueous formulationeffective to:

i) at least substantially kill or inactivate potentially-infectiouspathogens in the sample;

ii) lyse a portion of cells to release RNAs and/or DNAs from the sample;and

iii) substantially inhibit or prevent the released polynucleotides inthe sample from further hydrolysis or enzymatic degradation,modification, or inactivation, so as to obtain the population ofpolynucleotides from the sample.

Such sample may be of any origin, including, without limitation, aclinical or veterinary sample; an environmental or ecological sample, aforensic or crime scene sample, or such like, and may contain one ormore nucleic acids that are of viral, microbial, animal, or plantorigin, or any combination thereof.

Preferably, the methods of the invention will include at leastcontacting the sample with an amount of one or more of the disclosedcompositions at a temperature of from 0° C. to about 40° C. (morepreferably at a temperature of 4° C. to about 35° C., and still morepreferably at a temperature of 10° C. to about 30° C.) for a period oftime of at least 24 hrs, more preferably, for a period of time of atleast 48 hrs, at least 72 hrs, at least 96 hrs, or longer, withoutcausing substantial deterioration, degradation, enzymatic cleavage,and/or nucleolytic digestion, modification, or processing of the nucleicacids contained within a sample contacted with such a composition.

In certain embodiments, the methods of the invention will include atleast contacting the sample with an amount of one or more of thedisclosed compositions at a temperature from about 0° C. to about 40° C.(more preferably at a temperature from about 4° C. to about 35° C.,still more preferably at a temperature from about 10° C. to about 30°C., and more preferably still at a temperature from about 15° C. toabout 25° C.) for a period of time of at least 7 days, more preferably,for a period of time of at least 14 days, at least 21 days, at least 28days, or even longer without causing significant deterioration,degradation, enzymatic cleavage, and/or nucleolytic processing of thenucleic acids contained within a sample so processed. It should beunderstood that associating a sample with an inventive composition needonly occur for a short time, but to avoid the need for immediateseparation of the nucleic acids from the sample and the one-stepcomposition of the invention all the materials may remain in contact forthe time periods specified above without any substantial, or withoutany, degradation of the nucleic acids.

Preferably, the integrity of a population of polynucleotides releasedfrom the sample into the composition will be substantially maintained,even when the composition comprising the sample is stored at ambienttemperatures, and even for prolonged periods of time, including, withoutlimitation, storage for greater than about 10 days, greater than about20 days, or even greater than about 30 days or more. Likewise, it isdesirable that the integrity of a population of polynucleotides releasedfrom the sample into the composition will be substantially maintained,even when the composition comprising the sample is stored at subtropicaland tropical temperatures—even for prolonged periods of time, including,without limitation, storage for greater than about 5 days, greater thanabout 15 days, or even greater than about 25 days or more.

In the practice of the present methods, it is preferable that at leastone or more biological cells contained within the sample aresubstantially lysed to release at least a first population ofpolynucleotides contained within such cells into the composition.Preferably, the components of the disclosed composition are sufficientto release such a population from the remaining cellular debris(including, without limitation, lipids, proteins, polysaccharides,cellular components, and such like).

It is also desirable in the practice of the present methods that atleast one or more exogenous or endogenous nucleases that may be presentin, on, or about the sample itself, will be sufficiently inactivated byone or more components of the composition such that the resultingnucleic acids are not destroyed, damaged, or nucleolytically cleavedwhen the biological cells contained within the sample are substantiallylysed to release the population of polynucleotides from the cells.Preferably, one or more components of the disclosed composition areeffective to kill, inactivate, or substantially inhibit the biologicalactivity of a DNAse or an RNAse, when such a protein is present in thesample.

It is also desirable in the practice of the present methods that whenone or more microbes, viruses, and/or pathogens are present in, on, orabout the sample when collected, such microbes, viruses, and/orpathogens will be killed or sufficiently inactivated by one or morecomponents of the composition to facilitate safe handling of the sampleby the practitioner. Preferably, one or more components of the disclosedcomposition are effective to render a pathogenic sample substantially,or preferably entirely, non-pathogenic without the need for addingadditional components to the composition. However, in certainapplications, it may also be desirable to include one or more additionalanti-microbial, anti-viral, or anti-fungal agents to the compositions torender them substantially non-pathogenic, and thus, same for handling bythe practitioner.

Preferably, the composition containing the sample is at leastsufficiently stable, or is entirely stable, to permit storage of thesample in the composition at ambient temperature or colder at leastsubstantially (or entirely) from the time of collection to the time ofanalyzing a population of polynucleotides from the sample. As usedherein, “ambient temperature” can refer to temperatures of about 18° C.to 25° C., or in some embodiments about 20° C. to 22° C.

In certain embodiments, the composition containing the sample may bestored at a temperature of about 0° C. to about 40° C., more preferablyat a temperature of about 4° C. to about 30° C., more preferably, at atemperature of about 10° C. to about 25° C., at least substantially fromthe time of collection to the time that the polynucleotides obtainedfrom the sample are further isolated, purified, or characterized usingone or more conventional molecular biology methodologies.

In certain embodiments, the composition containing the sample suspectedof containing nucleic acids will stabilize the nucleic acids to theextent that they either remain at least substantially non-degraded(i.e., at least substantially stable) even upon prolonged storage of thecomposition at ambient, refrigerator, or sub-zero temperatures. It willbe desirable that this stability provides that at least about 70%, atleast about 85%, more preferably at least about 90%, more preferably atleast about 95%, or even more preferably, at least about 98% of thepolynucleotides contained within the stored sample will not be degradedupon prolonged storage of the sample. In certain embodiments,substantially all of the polynucleotides contained within the samplewill be stabilized such that the original integrity of thepolynucleotides is preserved during the collection, lysis, storage, andtransport of the processed sample.

In certain embodiments, the method will preferably provide a populationof nucleic acids prepared from a biological sample in which less thanabout 15% of the polynucleotides contained in the sample will bedegraded during the collection, lysis, storage, and transport of thesample after it has been stored in the composition at a temperature offrom −20° C. to about 40° C. for a period of at least 24, 48, 72, or 96hrs or longer after the sample was initially introduced into thecomposition.

In related embodiments, the method will preferably provide a populationof nucleic acids prepared from a biological sample in which less thanabout 10% of the polynucleotides contained in the sample will bedegraded during the collection, lysis, storage, and transport of thesample after it has been stored in the composition at a temperature offrom −20° C. to about 40° C. for a period of at least 24, 48, 72, or 96hrs or longer after the sample was initially introduced into thecomposition.

Likewise, in some applications of the methodology disclosed herein, useof the disclosed compositions will preferably provide a population ofnucleic acids that are prepared from a biological sample, wherein lessthan about 5% of the polynucleotides contained in the sample will bedegraded during the collection, lysis, storage, and transport of thesample after it has been stored in the composition at a temperature from−20° C. to about 40° C. for a period of at least 24, 48, 72, or 96 hrsor longer after the sample was initially introduced into thecomposition.

In some instances, the population of nucleic acids prepared by thepresent methods may be maintained with sufficient integrity such that nomore than about 1 or 2% of the sample will be degraded even when thecomposition is stored at a temperature from 0° C. to about 40° C. forperiods of several days to several weeks. In fact, the inventors haveshown that samples of nucleic acids isolated using the disclosed methodsremain at least substantially stable, preferably stable, in theirnon-degraded form for periods of several weeks to even several months ormore, even when the composition containing the nucleic acids is storedat a temperature from 10° C. to about 40° C. In one preferredembodiment, the upper limit on the above-noted temperature ranges isabout 37° C. Thus, the term “stable” as used herein may refer to thevarious embodiments noted above regarding the integrity of thepopulation of nucleic acids after a particular time lapse at a giventemperature.

Commercial Formulations and Kits

The present invention also provides kits and sample collection systemsutilizing the disclosed compositions and collection/storage/transportsolutions described herein. In particular embodiments, such samplecollection systems may include a collection device, such as a swab,curette, or culture loop; and a collection vessel, such as a vial testtube, or specimen cup, that contains one or more of the compositionsdisclosed herein. The collection vessel is preferably releasablyopenable, such that it can be opened to insert the one-step compositionsand closed and packaged, opened to insert the sample and optionally aportion of the collection device and closed for storage and transport,or both. The collection vessel may use any suitable releasably openablemechanism, including without limitation a screw cap, snap top,press-and-turn top, or the like. Such systems may also furtheroptionally include one or more additional reagents, storage devices,transport devices, and/or instructions for obtaining, collecting,lysing, storing, or transporting samples in such systems. In a preferredembodiment, the one-step compositions of the invention may already bedisposed in the reaction zone into which the sample may be associated.In such embodiments, the invention requires only a collection device andthe collection vessel.

The kit may also include one or more extraction devices to help liberateand separate the nucleic acids to obtain at least substantially pureRNA/DNA to be analyzed.

Kits may also be packaged for commercial distribution, and may furtheroptionally include one or more collection, delivery, transportation, orstorage devices for sample or specimen collection, handling, orprocessing. The container(s) for such kits may typically include atleast one vial, test tube, flask, bottle, specimen cup, or othercontainer, into which the composition(s) may be placed, and, preferably,suitably aliquotted for individual specimen collection, transport, andstorage. The kit may also include a larger container, such as a case,that includes the containers noted above, along with other equipment,instructions, and the like. The kit may also optionally include one ormore additional reagents, buffers, or compounds, and may also furtheroptionally include instructions for use of the kit in the collection ofa clinical, diagnostic, environmental, or forensic sample, as well asinstructions for the storage and transport of such a sample once placedin one or more of the disclosed compositions. The kit may include, e.g.,multiples of about 5 or more of the various collection devices andcollection vessels and any other components to be included, so that thekits can be used to collect multiple samples from the same source ordifferent sources.

DESCRIPTION OF THE DRAWINGS

For promoting an understanding of the principles of the invention,reference will now be made to the embodiments, or examples, illustratedin the drawings and specific language will be used to describe the same.It will nevertheless be understood that no limitation of the scope ofthe invention is thereby intended. Any alterations and furthermodifications in the described embodiments, and any further applicationsof the principles of the invention as described herein are contemplatedas would normally occur to one of ordinary skill in the art to which theinvention relates.

The following drawings form part of the present specification and areincluded to demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to the followingdescription taken in conjunction with the accompanying drawings, inwhich like reference numerals identify like elements, and in which:

FIG. 1 shows the extraction efficiency of PrimeStore™ (ver. 1).PrimeStore (ver. 1 [depicted here as “One-step+”]) compared to the LysisSolution provided in the RNaqueous®-Micro Kit (Ambion, Cat#AM1931) usinga standard amount of whole influenza A virus. For the comparison eitherthe one-step formulation or the Lysis Solution provided in the kit wasused for viral RNA lyses and then extracted according to manufacturerprotocols. Replicate reactions were processed and analyzed by real-timeRT-PCR (rRT-PCR) using an ABI 7500 sequence detection system;

FIG. 2 shows the extraction efficiency of PrimeStore™ Solution (ver. 1)compared to commercial kits. Homogenized cotton rat nose(*) challengedwith influenza A (H3N2) or a human clinical influenza A (H1N1) samplescollected during the 2006-07 season were lysed in the PrimeStore™Solution or lysed using the respective lyses solution, protocol, andextraction procedure from three commercially available kits:RNaqueous-Micro (Ambion Cat#AM1931), QiaAmp Viral Mini Kit (Qiagen), andAI/NCD MaxMag (Ambion) Kit. Extraction efficiency was evaluated usingthe ABI 7500 with the comparative CT method. The relative CT scores andviral copies detected were optimal when PrimeStore™ (depicted as the“one-step formulation”) was utilized in place of the respective lysesbuffer for each commercial kit;

FIG. 3 shows the preservation of naked RNA in PrimeStore™ Solution vs.Ambion RNA Storage Solution Single-stranded Avian H5 RNA was stored inPrimeStore™ solution, RNA storage solution (Ambion), or water at ambienttemperature (22-24° C.) for 96 hours. A total of 5 pg of RNA wasextracted using the RNaqueous®-Micro Kit (Ambion, Cat#AM1931) accordingto manufacturer recommendations and analyzed using real-time RT-PCR onan ABI 7500 (Applied Biosystems). Values are given as cycle thresholds(CT) using the absolute quantification method;

FIG. 4A shows an example of a PrimeStore™ packaging format for clinicaldiagnostic collection;

FIG. 4B Directions of sample collection using a clinical collection swab(Copan Diagnostics);

FIG. 4C a 5 mL collection tube containing 1.5 mL of PrimeStore™Solution;

FIG. 4D a schematic demonstration of the manipulation of the system;

FIG. 4E a schematic demonstration of the manipulation of the system;

FIG. 5 shows an exemplary commercial PrimeStore™ Collection Solution.Three exemplary commercial collection solution formats: 25 mL bottle,and the 5 mL and 1.5 mL tube formats;

FIG. 6 illustrates the ability of PrimeStore™ Solution to rapidly killmicroorganisms. Shown is a comparison of cell growth of MRSA either inculture medium (TSB), or in a solution of PS. After 10 seconds inPrimeStore™ Solution, no viable bacterial pathogens were detected.

FIG. 7A shows the inactivation of chicken cloacal specimens inPrimeStore™ Solution (Ver. 1). PrimeStore™ Solution inactivatesmicrobial agents in 1 hr. Four original chicken cloacal samples wereimmersed in PrimeStore™ Solution (top row) or water (bottom row) andsubsequently plated on blood agar plates;

FIG. 7B demonstrates that PrimeStore™ inhibits RNA base hydrolysis for30 days at room temperature. RNA was incubated at room temperature(22-26° C.) in PrimeStore™ (gel lane 1 and 3) and water (gel lane 2 and4), and subsequently RT-PCR amplified (1500 base pairs) at Day 0 and Day30. PrimeStore™ preserved collected RNA, and prevented RNA/DNAdegradation at room temperature up to 30 days;

FIG. 8A depicts the real-time RT-PCR analysis of “naked” influenza Aavian H5 RNA template preserved in PrimeStore™ Solution after incubationin RNA/DNA nucleases. H5 cRNA (2 ng) was incubated with ribonuclease Aand T1, and DNAseI for 1 hour @ 37° C. and extracted using theRNAaqueous®-Micro Kit (Ambion). Triplicate reactions were included foreach reaction condition. Real-time RT-PCR Cycle threshold (CT) values ofnaked RNA preserved in PrimeStore with added nucleases (average CT:22.88) were similar to an equal quantity of template cRNA control(average CT: 23.70). Template cRNA reactions subjected to nucleasedigestion without PrimeStore™ were almost completely degraded (averageCT 39.58);

FIG. 8B depicts the real-time RT-PCR analysis and gel electrophoresis of“naked” influenza A avian H5 RNA template preserved in PrimeStore™Solution after incubation in RNA/DNA nucleases @ 37° C. for 7 days. Twonanograms of H5 cRNA was incubated with RNase A and T1, and DNase I,then extracted using the RNAaqueous®-Micro Kit (Ambion) after 7 days.Duplicate reactions were included for each reaction. Real-time RT-PCRCycle Threshold (CT) values of naked RNA preserved in PrimeStore™ withadded nucleases (average CT: 33.51) were detected after 7 days. TemplatecRNA reactions subjected to nuclease digestion without PrimeStore werecompletely degraded and similar to NTC reactions.

FIG. 8C demonstrates that PrimeStore™ is impervious to nucleasedigestion. Gel electrophoresis of post-amplified product. Lane 3 is thePCR product from template RNA+PrimeStore™ at 37° C. for 7 days, and Lane5 amplification of positive control RNA. Lane 5 (no amplification) isRNA without PrimeStore™ Lane 2 and 6 are 100 bp ladder, and NTCreactions, respectively.

FIG. 9 illustrates that PrimeStore™ preservation is superior to othersolutions. PrimeStore™ (Ver. 2 and Ver. 2.2) Preservation of RNA frominfluenza A virus compared to Qiagen AVL buffer, ethanol, and ViralTransport Media (VTM) at ambient temperature (22-25° C.) for 30 days.

FIG. 10 shows the extraction efficiency of Influenza A virus preservedin PrimeStore™ (Ver. 2.2) for 30 days at various temperatures.Environmental (21-37° C.); Freeze-thawed (−25° C.; 32×); ambienttemperature (22-26° C.); and Lane 5: refrigerated (4° C.).

FIG. 11 is a graph of critical threshold vs. molar concentration usingwhole influenza A virus with TCEP as the reducing agent;

FIG. 12 is a graph of the critical threshold vs. molar concentrationusing H5N1 Avian influenza ssRNA with TCEP as the reducing agent;

FIG. 13A and FIG. 13B show the comparison between TCEP and β-ME asreducing agent components of the PrimeStore™ Solution compositions,using a water only control. In FIG. 13A, H5 avian influenza RNA wasemployed, while in FIG. 13B, whole virus were used.

FIG. 14A shows the results of a study employing PrimeStore™ solution inpreserving nucleic acids from blood. PrimeStore Extraction Efficiency ofwhole blood spiked with RNA compared to the lysis solution in theQIAamp® DNA Blood Mini Kit. 0.1 pg and 1 pg of influenza A RNA werespiked and extracted using PrimeStore™ or AL Lysis buffer. At both RNAconcentrations, PrimeStore™ produced superior results as evident byreal-time RT-PCR CT scores;

FIG. 14B tabulates data from the study shown in FIG. 14A involving theextraction of “naked” H5 avian influenza ssRNA from blood tubes.PrimeStore™ Extraction Efficiency of whole blood spiked with RNAcompared to the lysis solution in the QIAamp® DNA Blood Mini Kit usingdifferent blood anticoagulants. PrimeStore™ was superior compared to theAL Lysis Buffer from Qiagen using Blood spiked with RNA in commonanticoagulant blood-collection tubes; and

FIG. 14C tabulates data from the study shown in FIG. 14A involving thecomparison of in PrimeStore™ Solution vs. a commercial extraction kit(Qiagen). Shown is the Extraction Efficiency of PrimeStore™ for wholeblood spiked with RNA compared to the lysis solution in the QIAamp® DNABlood Mini Kit. 0.1 pg and 1 pg of Influenza A viral RNA were spiked andextracted using PrimeStore or AVL Lysis buffer. At both RNAconcentrations, PrimeStore™ produced superior results as evident byreal-time RT-PCR CT scores.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In theinterest of clarity, not all features of an actual implementation aredescribed in this specification. It will of course be appreciated thatin the development of any such actual embodiment, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which will vary from one implementation toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would be a routine undertakingfor those of ordinary skill in the art having the benefit of thisdisclosure.

The extended stabilization, collection, transport, and preservationimparted by the disclosed formulations are particularly advantageouswhen a sample or specimen is located in a geographical region that isremote from a testing facility. Remote locations, also referred to asfield sites, encompass a variety of environments where diagnostictesting is typically not performed. These sites include doctors'offices, triage centers, airports, border crossings, outbreak areas, anda variety of outdoor locations. The disclosed compositions and methodsfor their use offer particular advantages in locations where there is noaccess to electricity and/or refrigeration, or where access isinconsistent. Because of the extended stability at room temperature, asample can be taken from any remote location, for example withoutlimitation at a malarial outbreak site in Africa, and the sample can beshipped to the United States or Europe for diagnostic analysis in alaboratory. Because the disclosed collection formulations are stable atroom temperature or below, and preferably even at tropical orsubtropical temperatures for a time, they can routinely be taken intothe field without worry that the component reagents (such as RNAcontrols) themselves will degrade until a sample can be analyzed,typically at a remote location from the collection.

The compositions of the invention may be any suitable aqueousformulation as described herein, including but not limited to asolution, suspension (incl. colloidal suspension), slurry, emulsion,homogenate, or the like. A preferred aqueous formulation is a solution,and therefore the term “solution” has been used in the exemplary sensethroughout the detailed description of the preferred embodiments torefer to any of the aqueous compositions of the invention.

Specimen Collection for Clinical Diagnostic Laboratories

Collection is first step in diagnostic platforms or molecular protocolsrequiring the detection of potentially minute amounts of nucleic acidsfrom pathogens including viruses. To facilitate the dynamic advancementsin nucleic acid based detection strategies and their integration intothe mainstream diagnostic laboratories there is a colossal need forreliable, robust, and standardized collection systems developedspecifically with the intent of being utilized for downstream nucleicacid based detection such as the aforementioned platforms. The inventioncan alternatively be adapted for transport of nucleic acids from adoctor's office or operating room, or alternatively transported to aregional center, such as a hospital.

A clinical or veterinary specimen or a forensic or environmental samplecollection system may include one or more collection tools and one ormore reagents for efficiently: 1) obtaining a high yield of suitablespecimen beyond what is currently available in the art; 2) inactivatingpotentially infectious biological pathogens so that they are no longerviable and can be handled; shipped, or transported with minimal fear ofpathogen release or contamination; or 3) effectively stabilizing andpreserving lysed ‘naked’ RNA/DNA polymers from hydrolysis or nucleasedegradation for prolonged periods at ambient temperatures until samplescan be processed at a diagnostic laboratory, and preferably forachieving two or more, or all three, of these goals.

The collection/transport solutions of the present invention can providea number of improvements and benefits over those presently available inthe art. Exemplary benefits include, without limitation, one or more ofthe following:

Inactivation, killing, and/or lysis of microbes, viruses, or pathogens;

Destruction and/or inactivation of exogenous or endogenous nucleases,including, without limitation, RNase and/or DNase;

Compatibility with a variety of conventional nucleic acid extraction,purification, and amplification systems;

Preservation of RNA and/or DNA integrity within the sample;

Facilitation of transport and shipping at ambient temperatures, evenover extended periods of time, or extreme temperature variations; and

Suitability for short- (several hours to several days), intermediate-(several days to several weeks), or long- (several weeks to severalmonths) term storage of the isolated nucleic acids.

The disclosed compositions are particularly well suited forpoint-of-care, field studies, in-home health care or testing,triage/emergency and casualty assessment(s), mobile forensics,pathology, epidemiological sampling, crime scene investigation,paternity testing, pre- and post-pregnancy genetic screening,rape/incest testing and family counseling, confidential screening andtesting for sexually transmitted diseases, including, withoutlimitation, HIV, syphilis, Chlamydia, gonorrhoeae, or other venerealdiseases and the like, and may be of particular value during themonitoring, etiology, and control of epidemic or pandemic diseases inboth human and animal populations domestically and abroad. Thecompositions may be of particular relevance in collecting and analyzingInfluenzavirus samples, including without limitation to predict and helpmanage shift and drift and to manage an imminent or ongoing pandemic.

In certain embodiments, the nucleic acid(s) isolated by the methods ofthe present invention may serve as a template in one or more subsequentmolecular biological applications, assays, or techniques, including,without limitation, genetic fingerprinting; amplified fragment lengthpolymorphism (AFLP) polymerase chain reaction (PCR); restrictionfragment length polymorphism analysis (RFLP); allele-specificoligonucleotide analysis (ASOA); microsatellite analysis; Southernhybridization; Northern hybridization; variable number of tandem repeats(VNTR) PCR; dot-blot hybridization; quantitative real-time PCR;polymerase cycling assembly (PCA); nested PCR; quantitative PCR (Q-PCR);asymmetric PCR; DNA footprinting; single nucleotide polymorphism (SNP)genotyping; reverse transcription PCR (RT-PCR); multiplex PCR (m-PCR);multiplex ligation-dependent probe amplification (MLPA);ligation-mediated PCR (LmPCR); methylation specific PCR (MPCR);helicase-dependent amplification (HDA); overlap-extension PCR (OE-PCR);whole-genome amplification (WGA); plasmid isolation; allelicamplification; site-directed mutagenesis; high-throughput geneticscreening; or the like, or any combination thereof.

The compositions of the present invention provide clinical/environmentalcollection solutions that efficiently achieve at least three, andpreferably all four of the following: 1) kill or inactivatepotentially-infectious pathogens, so that they are non-viable and can besafely handled, shipped or transported; 2) lyse cells to release RNAsand/or DNAs from the biological specimen contained in the collectionsystem; 3) protect the released or ‘naked’ polynucleotides in the samplefrom further hydrolysis or enzymatic degradation, modification, orinactivation; and 4) prolong the conventional time-frame for storage andtransportation of the processed sample under a variety of ambient, sub-or supra-optimal temperature conditions to maintain the fidelity andintegrity of the released polynucleotides until the biological materialcan be further processed or analyzed at a diagnostic facility oranalytical laboratory.

In one exemplary embodiment, the methods and formulations maintain atleast substantial stability of the nucleic acids in the sample for anextended period of time, e.g., for up to about 15 days, preferably up toabout 30 days, or more preferably up to about 60 days or more, withoutrefrigeration or freezing of the sample, and even when stored at roomtemperature, or ambient environmental conditions including those oftemperate, sub-tropical or tropical climates and the like. In otherembodiments, use of the disclosed compositions to prepare nucleic acidsfrom a sample of biological origin is desirable to maintain at leastsubstantial integrity and fidelity of the nucleic acids released fromthe sample for extended periods including, without limitation, at leastabout 5 to about 15 days, preferably at least about 10 to 20 days, morepreferably at least about 15 to 25 days, or more preferably still, atleast about 20 to 30 days or more, without a requirement forrefrigerating or freezing the sample either at the time of samplecollection or until the sample is further processed (or both) hours,days, weeks, or even months after originally being collected and placedinto the disclosed storage/collection/transport/stabilizationformulations.

Nucleic acids obtained from biological samples in the practice of thedisclosed methods are advantageously compatible with a number ofconventional molecular and diagnostic isolation, purification,detection, and/or analytic methodologies. The disclosed compositionsfacilitate recovery storage, and transport of populations of stabilized,substantially non-degraded, polynucleotides for use in a variety ofsubsequent methodologies, including, without limitation, nucleic acidisolation, purification, amplification, and molecular analytical and/ordiagnostic testing, assay, analysis, or characterization, and the like.

Exemplary Commercial Kits of the Present Invention

The following outline provides exemplary commercial kits employing thePrimeStore™ compositions of the present invention (FIG. 5).

Peel-Pouch Collection System

Five-mL tube containing 1.5 mL PrimeStore™ Solution; Collection swab(e.g., FlockedSWABS® [Copan Diagnostics, Inc., Murrieta, Calif., USA]);and instructions for collection and/or processing of samples. (packed,e.g., in 50 pouches/unit) (See FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, andFIG. 4E for a schematic demonstration of such systems).

PrimeStore™ Stock Solution (e.g., 25-mL Bottles)

Once formulated, PrimeStore™ stock solution is stable at 4° C. or belowfor periods of at least one year or more. Formulated PrimeStore™Solution has also been shown to be stable at ambient temperature (e.g.,about 20-30° C.) for periods of three to six months or more.

Once a sample is contacted with a PrimeStore™ formulation as disclosedherein, it can reasonably expected to be stored indefinitely attemperatures of 0° C. or below, at least one year or more underrefrigeration (e.g., ≈4° C. and at least 30 days or more at ambienttemperature (e.g., about 20-30° C.), without significant loss of nucleicacid composition, fidelity or integrity of the sample. For example,without limitation, the integrity of a population of polynucleotidesobtained from the sample is at least substantially maintained, andpreferably entirely maintained without detectable degradation, when thecomposition comprising the sample is stored at a temperature of fromabout −20° C. to about 40° C., for a period of from about 30 days toabout 60 days.

The kit may also include one or more vials including the inventivecompositions and one or more extraction devices to help liberate andseparate the nucleic acids to obtain at least substantially pure RNA/DNAto be analyzed.

Environmental Sample and Storage Systems

0.1-, 0.2-, 0.5-, 1-, 2-, or 3-mL collection vials each containing 0.1mL, 0.2 mL, 0.25 mL, 0.5 mL, 0.75 mL, or 1 mL PrimeStore™ solution; andinstructions for collection and/or processing of samples (packed, e.g.,10 vials/unit). The collection vials may be sized larger as neededdepending on the proposed collection method.

Definitions

The terms “about” and “approximately” as used herein, areinterchangeable, and should generally be understood to refer to a rangeof numbers around a given number, as well as to all numbers in a recitedrange of numbers (e.g., “about 5 to 15” means “about 5 to about 15”unless otherwise stated). Moreover, all numerical ranges herein shouldbe understood to include each whole integer within the range.

As used herein, the term “carrier” is intended to include anysolvent(s), dispersion medium, coating(s), diluent(s), buffer(s),isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) orsuch like, or a combination thereof that is pharmaceutically acceptablefor administration to the relevant animal or acceptable for a diagnosticpurpose, as applicable. The use of one or more delivery vehicles forchemical compounds in general, and peptides and epitopes in particular,is well known to those of ordinary skill in the pharmaceutical arts.Except insofar as any conventional media or agent is incompatible withthe active ingredient, its use in the diagnostic, prophylactic, andtherapeutic compositions is contemplated. One or more supplementaryactive ingredient(s) may also be incorporated into, or administered inassociation with, one or more of the disclosed immunogenic compositions.

As used herein, the term “nucleic acid” includes one or more types of:polydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other type ofpolynucleotide that is an N-glycoside of a purine or pyrimidine base, ormodified purine or pyrimidine bases (including abasic sites). The term“nucleic acid,” as used herein, also includes polymers ofribonucleosides or deoxyribonucleosides that are covalently bonded,typically by phosphodiester linkages between subunits, but in some casesby phosphorothioates, methylphosphonates, and the like. “Nucleic acids”include single- and double-stranded DNA, as well as single- anddouble-stranded RNA. Exemplary nucleic acids include, withoutlimitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), smallinterfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA(snRNA), and small temporal RNA (stRNA), and the like, and anycombination thereof.

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably, and include molecules that include at least oneamide bond linking two or more amino acid residues together. Althoughused interchangeably, in general, a peptide is a relatively short (e.g.,from 2 to about 100 amino acid residues in length) molecule, while aprotein or a polypeptide is a relatively longer polymer (e.g., 100 ormore residues in length). However, unless specifically defined by achain length, the terms peptide, polypeptide, and protein are usedinterchangeably.

As used herein, “sample” includes anything containing or presumed tocontain a substance of interest. It thus may be a composition of mattercontaining nucleic acid, protein, or another biomolecule of interest.The term “sample” can thus encompass a solution, cell, tissue, orpopulation of one of more of the same that includes a population ofnucleic acids (genomic DNA, cDNA, RNA, protein, other cellularmolecules, etc.). The terms “nucleic acid source,” “sample,” and“specimen” are used interchangeably herein in a broad sense, and areintended to encompass a variety of biological sources that containnucleic acids, protein, one or more other biomolecules of interest, orany combination thereof. Exemplary biological samples include, but arenot limited to, whole blood, plasma, serum, sputum, urine, stool, whiteblood cells, red blood cells, buffy coat, swabs (including, withoutlimitation, buccal swabs, throat swabs, vaginal swabs, urethral swabs,cervical swabs, rectal swabs, lesion swabs, abscess swabs,nasopharyngeal swabs, and the like), urine, stool, sputum, tears, mucus,saliva, semen, vaginal fluids, lymphatic fluid, amniotic fluid, spinalor cerebrospinal fluid, peritoneal effusions, pleural effusions,exudates, punctates, epithelial smears, biopsies, bone marrow samples,fluid from cysts or abcess contents, synovial fluid, vitreous or aqueoushumor, eye washes or aspirates, pulmonary lavage or lung aspirates, andorgans and tissues, including but not limited to, liver, spleen, kidney,lung, intestine, brain, heart, muscle, pancreas, and the like, and anycombination thereof. In some embodiments, the sample may be, or be from,an organism that acts as a vector, such as a mosquito, or tick, or otherinsect(s).

Tissue culture cells, including explanted material, primary cells,secondary cell lines, and the like, as well as lysates, homogenates,extracts, or materials obtained from any cells, are also within themeaning of the term “biological sample,” as used herein. Microorganisms(including, without limitation, prokaryotes such as the archaebacteriaand eubacteria; cyanobacteria; fungi, yeasts, molds, actinomycetes;spirochetes, and mycoplasmas); viruses (including, without limitationthe Orthohepadnaviruses [including, e.g., hepatitis A, B, and Cviruses], human papillomavirus, Flaviviruses [including, e.g., Denguevirus], Lyssaviruses [including, e.g., rabies virus], Morbilliviruses[including, e.g., measles virus], Simplexviruses [including, e.g.,herpes simplex virus], Polyomaviruses, Rubulaviruses [including, e.g.,mumps virus], Rubiviruses [including, e.g., rubella virus],Varicellovirus [including, e.g., chickenpox virus], rotavirus,coronavirus, cytomegalovirus, adenovirus, adeno-associated virus,baculovirus, parvovirus, retrovirus, vaccinia, poxvirus, and the like),algae, protozoans, protists, plants, bryophytes, and the like, and anycombination of any of the foregoing, that may be present on or in abiological sample are also within the scope of the invention, as are anymaterials obtained from clinical or forensic settings that contain oneor more nucleic acids are also within the scope of the invention. Theordinary-skilled artisan will also appreciate that lysates, extracts, ormaterials obtained from any of the above exemplary biological samplesare also within the scope of the invention.

As used herein, the term “buffer” includes one or more compositions, oraqueous solutions thereof, that resist fluctuation in the pH when anacid or an alkali is added to the solution or composition that includesthe buffer. This resistance to pH change is due to the bufferingproperties of such solutions, and may be a function of one or morespecific compounds included in the composition. Thus, solutions or othercompositions exhibiting buffering activity are referred to as buffers orbuffer solutions. Buffers generally do not have an unlimited ability tomaintain the pH of a solution or composition; rather, they are typicallyable to maintain the pH within certain ranges, for example from a pH ofabout 5 to 7.

As used herein, the term “biological molecule” refers to any moleculefound within a cell or produced by a living organism, including viruses.This may include, but is not limited to, nucleic acids, proteins,carbohydrates, and lipids. As used herein, a “cell” refers to thesmallest structural unit of an organism that is capable of independentfunctioning and is comprised of cytoplasm and various organellessurrounded by a cell membrane. This may include, but is not limited to,cells that function independently such as bacteria and protists, orcells that live within a larger organism such as leukocytes anderythrocytes. As defined herein, a cell may not have a nucleus, such asa mature human red blood cell.

Samples in the practice of the invention can be used fresh, or can beused after being stored for a period of time, or for an extended periodof time, including for example, cryopreserved samples and the like, andmay include material of clinical, veterinary, environmental or forensicorigin, may be isolated from food, beverages, feedstocks, potable watersources, wastewater streams, industrial waste or effluents, naturalwater sources, soil, airborne sources, pandemic or epidemic populations,epidemiological samples, research materials, pathology specimens,suspected bioterrorism agents, crime scene evidence, and the like.

As used herein, the term “patient” (also interchangeably referred to as“host” or “subject”) refers to any host that can serve as a source ofone or more of the biological samples or specimens as discussed herein.In certain aspects, the donor will be a vertebrate animal, which isintended to denote any animal species (and preferably, a mammalianspecies such as a human being). In certain embodiments, a “patient”refers to any animal host, including but not limited to, human andnon-human primates, avians, reptiles, amphibians, bovines, canines,caprines, cavines, corvines, epines, equines, felines, hircines,lapines, leporines, lupines, murines, ovines, porcines, racines,vulpines, and the like, including, without limitation, domesticatedlivestock, herding or migratory animals or birds, exotics or zoologicalspecimens, as well as companion animals, pets, and any animal under thecare of a veterinary practitioner. The invention may also be used tomonitor disease outbreak, progression, and epidemiological statisticsfor a variety of global populations, including, without limitation,wasting disease in ungulates, tuberculosis, ebola, SARS, and avianinfluenzas. In certain embodiments, the samples will preferably be ofmammalian origin, and more preferably of human origin.

The term “chaotrope” or “chaotropic agent” as used herein, includessubstances that cause disorder in a protein or nucleic acid by, forexample, but not limited to, altering the secondary, tertiary, orquaternary structure of a protein or a nucleic acid while leaving theprimary structure intact.

The term “e.g.,” as used herein, is used merely by way of example,without limitation intended, and should not be construed as referringonly those items explicitly enumerated in the specification.

The term “substantially free” or “essentially free,” as used herein,typically means that a composition contains less than about 10 weightpercent, preferably less than about 5 weight percent, and morepreferably less than about 1 weight percent of a compound. In apreferred embodiment, these terms refer to less than about 0.5 weightpercent, more preferably less than about 0.1 weight percent or even lessthan about 0.01 weight percent. The terms encompass a composition beingentirely free of a compound or other stated property, as well. Withrespect to degradation or deterioration, the term “substantial” may alsorefer to the above-noted weight percentages, such that preventingsubstantial degradation would refer to less than about 15 weightpercent, less than about 10 weight percent, preferably less than about 5weight percent, etc., being lost to degradation. In other embodiments,these terms refer to mere percentages rather than weight percentages,such as with respect to the term “substantially non-pathogenic” wherethe term “substantially” refers to leaving less than about 10 percent,less than about 5 percent, etc., of the pathogenic activity.

In accordance with long standing patent law convention, the words “a”and “an” when used in this application, including the claims, denotes“one or more.”

EXAMPLES

The following examples are included to demonstrate illustrativeembodiments of the invention. It should be appreciated by those ofordinary skill in the art that the techniques disclosed in the examplesthat follow represent techniques discovered to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of ordinary skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

Example 1—Formulation of Exemplary Storage Solutions

The present example provides a general formulation of the PrimeStore™(PanFlu) compositions of the present invention. Exemplary formulationsare also detailed in Examples 2-5.

Materials

Guanidine thiocyanate, sodium citrate, Antifoam A® Concentrate, andN-lauroylsarcosine, sodium salt, were all purchased from Sigma ChemicalCo. (St. Louis, Mo., USA). Tris(2-carboxyethyl) phosphine hydrochloride(TCEP) was obtained from Soltec Ventures Inc. (Beverly, Mass., USA).2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS) was obtained fromApplied Biosystems/Ambion (Austin, Tex., USA).2-[2-(Bis(carboxymethyl)amino)ethyl-(carboxymethyl)amino]acetic acid(EDTA) GIBCO® Ultra Pure was obtained from Invitrogen Corp. (Carlsbad,Calif., USA). All other reagents are available commercially fromSigma-Aldrich or USB Corporation.

TABLE 1 FORMULATION RANGES OF EXEMPLARY COMPONENTS FOR THE PREPARATIONOF PRIMESTORE ™ COMPOSITIONS REAGENT COMPONENT FINAL CONCENTRATIONRANGES 1. A chaotrope, e.g.: Guanidine thiocyanate about 0.5M to about6M or Guanidine hydrochloride about 0.5M to about 6M or Guanidineisocyanate about 0.5M to about 6M 2. An anionic detergent, e.g.:N-lauroyl sarcosine (inter alia Na salt) about 0.15% to about 1%(wt./vol.) or Sodium dodecyl sulfate, Same Lithium dodecyl sulfate, SameSodium glycocholate, Same Sodium deoxycholate, Same Sodiumtaurodeoxycholate, or Same Sodium cholate about 0.1% to about 1%(wt./vol.) 3. A reducing agent, e.g.: TCEP about 0.5 mM to about 30 mMor β-ME, DTT, formamide, or DMSO about 0.05M to about 0.3M 4. Achelator, e.g.: Sodium citrate about 0.5 mM to about 50 mM or EDTA,EGTA, HEDTA, DTPA, NTA, or APCA about 0.01 mM to about 1 mM 5. A buffer(e.g., TRIS, HEPES, MOPS, MES, Bis-Tris, etc.) about 1 mM to about 1M 6.An acid (e.g., HCl or citric acid) q.s. to adjust to a pH of about 6 to7, preferably 6.4 to 6.8 7. Nuclease-free water q.s. to desired finalvolume Optionally one or more of: 8. A surfactant/defoaming agent, e.g.:Antifoam A ® or Tween ® about 0.0001% to about 0.3% (wt./vol.) 9. Analkanol (e.g., methanol, ethanol, propanol, etc.) about 1% to about 25%(vol./vol.) 10. RNA or DNA about 1 pg to about 1 μg/mL

Example 2—Formulation of an Exemplary Storage Solution

The present example describes a first exemplary formulation of thecompositions of the invention. This formulation has also beenalternatively referred to by the inventors as “PrimeStore™ Solution” or“PSS” version 1.

TABLE 2 PREPARATION OF PRIMESTORE ™ COMPOSITION (VER. 1) Reagent FinalConcentration Guanidine thiocyanate 4M Sodium citrate 30 mM Sodiumdodecyl sulfate 0.25% (wt./vol.) N-lauroyl sarcosine, sodium salt 0.25%(wt./vol.) 2-mercaptoethanol (β-ME) 0.1M Antifoam A 0.1% (wt./vol.)Citric acid q.s. to adjust pH to 6.5 Nuclease-free water 11.82 mL

Example 3—Preparation of a Second Exemplary Storage Solution

The present example describes the preparation of another exemplarystorage solution according to the present invention. This formulationhas also been alternatively referred to by the inventors as PrimeStore™version 2.

TABLE 3 PREPARATION OF PRIMESTORE ™ COMPOSITION (VER. 2) FINAL REAGENTQUANTITY CONCENTRATION Guanidine thiocyanate 35.488 gm 3M TCEP 0.02867gm  1 mM Sodium citrate 0.2931 gm 10 mM N-lauroyl sarcosine, 0.5 gm 0.5% sodium salt (NLS) Antifoam A 200 μL 0.002% (10% solution) TRIS(1M) 10 mL 100 mM  EDTA (0.5M) 20 μL 0.1 mM  Hydrochloric acid (HCl)q.s. to adjust pH to 6.7 — Nuclease-free water q.s. to 100 mL —

Example 4—Preparation of a Third Exemplary Storage Solution

The present example describes the preparation of another exemplarystorage solution according to the present invention. This formulationhas also been alternatively referred to by the inventors as PrimeStore™version 2.2.

TABLE 4 PREPARATION OF PRIMESTORE ™ COMPOSITION (VER. 2.2) FINAL REAGENTQUANTITY CONCENTRATION Guanidine thiocyanate 35.488 gm 3M TCEP 0.02867gm  1 mM Sodium citrate 0.2931 gm 10 mM N-lauroyl sarcosine, 0.5 gm 0.5% sodium salt (NLS) Antifoam A 200 μL 0.002% (10% solution) TRIS(1M) 10 mL 100 mM  EDTA (0.5M) 20 μL 0.1 mM  Ethanol, molecular grade 23mL 23% (vol./vol.) (96-100%) Hydrochloric acid (HCl) q.s. to adjust pHto 6.7 — Nuclease-free water q.s. to 100 mL —Exemplary Protocol for Preparation of PrimeStore Solution (Ver. 2.2)

-   -   1. Add 40 mL of nuclease-free water to a clean beaker with a        stir bar.    -   2. Place beaker on a hot plate/stirrer and adjust temperature to        60-65° C. Set stirring speed to medium.    -   3. Add 35.488 gm of guanidine thiocyanate slowly to the water        allowing it to dissolve as added.    -   4. Add 0.0287 gm of TCEP to beaker and increase stirrer speed to        help dissolve crystals.    -   5. Add 0.2931 gm of sodium citrate to the beaker.    -   6. Add 0.5 gm of NLS to the solution. Increase stirrer speed to        create a vortex in the beaker. This will pull the NLS into the        solution and help dissolve the reagent.    -   7. Vortex a prepared 10% Antifoam A solution (1 mL Antifoam A        Concentrate+9 mL nuclease-free water). Pipette 200 μL of the 10%        Antifoam A into the solution.    -   8. Pipette 10 mL of 1 M TRIS into the solution.    -   9. Pipette 20 μL of 0.5 M EDTA into the solution.    -   10. Increase the temperature to bring the solution to 75-80° C.        and stir for 3-5 minutes.    -   11. Remove beaker from heat and allow solution to cool to room        temperature (≈22-25° C.).    -   12. Add 23 mL of ethanol to the solution and mix thoroughly.    -   13. Adjust pH to 6.7 with HCl.    -   14. Pour solution into a clean 100 mL graduated cylinder.    -   15. Add nuclease-free water to bring total volume to 100 mL.    -   16. Transfer solution to a labeled sterile container. Store at        room temperature (≈22-25° C.).        -   *Note: Preferably, make sure each reagent is completely            dissolved before adding the next.

Example 5—Comparison of PrimeStore™ Solutions to ConventionalFormulations

A sample of homogenized nasal tissue from a cotton rat (Sigmodonhispidus) challenged with influenza A (H3N2) or a human clinicalinfluenza A (H1N1) sample collected as a human clinical nasal washduring the 2006-07 season were placed in PrimeStore™ Solution (Ver. 1)and tested compared to the respective lysis formulation and protocol,and extraction procedure, from three commercially available kits:RNAqueous®-Micro (Ambion, Austin, Tex., USA), QIAamp Viral RNA Mini Kit(Catalogue #52904, Qiagen, Valencia, Calif., USA), and MagMax AI/NDViral RNA Isolation Kit (Catalogue #AM1929, Ambion). Extractionefficiency was evaluated using the ABI 7500 sequence detection systemwith the comparative threshold cycle (C_(T)) method (See FIG. 2). InFIG. 2, “delta Rn” represents the fluorescent reporter signal minus abaseline amount. As shown in FIG. 1 and FIG. 2, the relative C_(T)scores and viral copies detected were optimal when the fixingformulation was used in place of the respective conventional lysisbuffer for each commercial kit. In these two sample types, thecompositions of the invention worked better than the two conventionalKits for extraction purposes. The PrimeStore™ Solution (ver. 1)composition was also shown to be readily compatible with commerciallyavailable nucleic acid extraction kits. FIG. 1 illustrates RNAextraction results where the version 1 of PrimeStore™ Solution was usedin conjunction with three commercially available kits: Qiagen ViralMini, Ambion RNAqueous Mini, and Ambion Al/NCD MagMax. As illustrated byFIG. 1, when the lysis buffer of the extraction kit was replaced withthe fixing formulation (denoted on the figure as “One-Step+), superiornucleic acid extraction was achieved when compared to extraction usingkits according to standard protocol (denoted on the figure as“One-Step-”. Extraction efficiency was measured by real time (r) reversetranscription (RT) polymerase chain reaction (PCR) [rRT-PCR].

FIG. 3 shows preservation of naked RNA in PrimeStore™ Solution comparedto preservation in a prior solution, with water used as a control. Asillustrated in FIG. 3, detection (by fluorescence) occurred at theearliest amplification cycle for RNA stored in PrimeStore™ Solution(ver. 1) at all time-points assayed.

Example 6—PrimeStore™ Solution for the Collection of Nasal WashSpecimens

A prospective clinical detection study was conducted using nasal washspecimens from: 1) symptomatic pediatric patients and 2) asymptomatic orsymptomatic family members. Detection of influenza virus compared nasalwash specimens collected in PrimeStore™ Solution and Viral TransportMedium (VTM) by real-time RT-PCR (rRT-PCR) and traditional culture,respectively. Genetic characterization of influenza genes encodinghemagglutinin (HA), neuraminidase (NA), and matrix surface (MA) proteinswere performed using select nasal wash specimens preserved inPrimeStore™ Solution to evaluate vaccine effectiveness and drugsensitivity within viral strains.

Influenza is a highly evolving, RNA-based respiratory virus responsiblefor more than 200,000 hospitalizations and about 36,000 fatalities eachyear in the United States. Widespread emergence of influenza driftvariants among contemporary circulating human viruses prompted a changein all three vaccine components for the upcoming 2008/09 season.Increased morbidity and mortality during the 2007/08 season included 72influenza-associated pediatric deaths and continued drug resistance(oseltamivir [TamiFlu®, Roche Laboratories, Inc., Nutley, N.J., USA] andadamantadine) within circulating strains.

Materials and Methods

A total of 100 pediatric (index) patients who met the clinical casecriteria for influenza infection and 126 family contacts were enrolledin the study. Nasal washings were placed into PrimeStore™ Solution andUniversal Viral Transport Medium (Becton-Dickinson, Franklin Lakes,N.J., USA) and analyzed by rRT-PCR or culture analysis, respectively.rRT-PCR was performed using influenza type (A or B) and subtype (H3, H1,H5) specific primers/probes according to Daum et al. (2007). Furthergenetic characterization of selected clinical samples preserved inPrimeStore™ Solution was performed using standard RT-PCR and directnucleotide sequencing of the hemagglutinin HA, NA, and MA viralproteins.

Results

Of the total samples evaluated (N=226; 100 index, 126 family contacts),66 (29%) tested positive for influenza virus (45 H3N2, 2 H1N1 and 19 B)by rRT-PCR. rRT-PCR from nasal washings preserved in PrimeStore™Solution detected influenza virus from 11 patients (9 Flu A and 2 Flu B)that were not detected by culture (Table 5 and Table 6). Of these 11specimens, five were from patients enrolled as family contacts.

Phylogenetic analysis of influenza A and B HA genes exhibited driftingcompared to the 2007/08 vaccine strains and revealed a higher genetichomology to the 2008/09 Brisbane vaccine strains. Some geneticdifferences in viruses were noted among family members, particularlyamong influenza A (H3N2) strains. MA analysis revealed adamantaneresistance in all influenza A H3N2 strains, but sensitivity in both H1N1viruses. All influenza B strains (n=18) were sensitive to theneuraminidase inhibitor drugs zanamivir (Relenza® GlaxoSmithKline,Research Triangle Park, N.C., USA) and oseltamivir (Tamiflu® Roche)based on the presence of an aspartic acid (D) at amino acid 197(influenza B numbering) in the NA gene.

Real-Time Rt-PCR Vs. Culture

rRT-PCR is superior to traditional culture for the detection ofinfluenza virus from original nasal wash specimens preserved inPrimeStore™ solutions: influenza was detected within 2 hours (c.f. 2 to7 days for conventional culture methods); and the analyses were moresensitive (11 specimens; 9 Flu A and 2 Flu B detected below culturelimits). Moreover, the use of molecular diagnostic methods in lieu ofconventional organism culture did not propagate potentially infectiousviruses, and simultaneously provided the type and subtype of theinfluenza virus.

Genetic Analysis

Vaccine Relatedness

H3N2 Strains: Analysis of the HA1 gene of the influenza A (H3N2)hemagglutinin (HA) revealed genetic drift including five amino aciddifferences in all Texas strains compared to the 2007-08A/Wisconsin/67/2005 vaccine strain. One HA1 mutation noted in all theTexas strains (D122N) is a potential glycosylation site. All A/Texas(H3N2) strains exhibited a greater HA homology (99.0-99.7%) to the newlyselected 2008-09 A/Brisbane/10/2007 strain.

H1N1 Strains:

The hemagglutinin HA1 gene of the 2 influenza A (H1N1) exhibited 7 aminoacid changes compared to A/Solomon Island/3/2006 vaccine strain. Foursubstitutions (R90K, T145V, K210T and E290K) were within known H1antibody combining sites. Both Texas H1N1 strains exhibited greater HAhomology (98.8% and 99.4%) to the newly selected 2008-09A/Brisbane/59/2007 vaccine strain.

Influenza B Strains:

Analysis of the HA1 hemagglutinin and neuraminidase genes revealed allTexas strains were of the B/Yamagata lineage and genetically morehomologous to the 2008-09 B/Brisbane/5/2007 vaccine strain than the2007/08 B/Malaysia/2506/2004 vaccine strain.

Family Mutation

Amino acid changes were noted in the NA, HAL M1 and M2e among familymembers. The HA1 Hemagglutinin showed the highest mutation of theinfluenza genes analyzed, with one family exhibiting five amino acidchanges.

Analysis of the highly conserved 24 amino acid M2e viral surface protonpump showed some variation within families. One index patient straincontained 3 unique amino acid M2e substitutions that were ‘wild-type’within family member strains.

Antiviral Susceptibility

Adamantane:

Matrix gene (MA) genetic analysis, specifically a serine-to-asparaginesubstitution at position 31 (S31N), revealed adamantane resistance inall influenza A (H3N2) strains but sensitivity in both influenza A(H1N1) viruses.

Neuraminidase Inhibitors:

All Texas influenza A (H3N2) isolates were shown to be sensitive tooseltamivir through genetic analysis of E119V, R292K, and N294Ssubstitutions in the NA gene. Genetic analysis of the influenza B NAgene revealed that all Texas strains contained an aspartic acid (D)residue at position 197, and are thus likely sensitive to oseltamivir.

The protocols and tests herein can be adapted for other microbes liketuberculosis, malaria, staphylococcus, and the like and other pathogenswhere there is a need to know antimicrobial susceptibility quickly.

Example 7—Influenza Sample Collection Using PrimeStore™ Solution

The compositions of the present invention provide a single samplecollection, transport, and storage reagent that facilitate: 1) procuringhigh quality nucleic acids from clinical or environmental specimens, 2)inactivation of potentially infectious biological pathogens for safehandling and transport of specimens, and 3) stabilization andpreservation of released ‘naked’ RNA/DNA preventing hydrolysis/nucleasedegradation for prolonged periods at ambient temperatures. The resultsof one such study are presented in the following example:

TABLE 5 Influenza Subtype Detection: rRT-PCR vs. Culture Influenza AInfluenza B Total FluA Samples rRT-PCR Culture rRT-PCR Culture TotalFluB Samples (N = 47) (N = 47) (N = 40) (N = 19) (N = 17) (N = 19) IndexPatients (28) 28/28 (100%) 23/28 (82%) 16/16 (100%) 14/16 (86%) IndexPatients (16) Family Contacts (19) 19/19 (100%) 15/19 (79%)  3/3 (100%) 3/3 (100%) Family Contacts (3)

TABLE 6 Positive Influenza Detection: rRT-PCR vs. Culture Total SamplesFlu Positive rRT-PCR (N = 226) (N = 66) (N = 66) Culture (N = 66) IndexPatients (100) 44/100 39/39 (100%) 37/39 (94%) Family Contacts (126)22/126 27/27 (100%) 18/27 (67%)

This example illustrates the effectiveness of the PrimeStore™ Solution(ver. 2.2) in killing pathogenic microbe(s).

Methods

Real-time RT-PCR was used to assay influenza A (H5N1) virus nucleic acidpreserved in PrimeStore™ Solution. A time-course study at roomtemperature was carried out to evaluate the integrity of clinicalspecimens, cloacal samples, and cloned template avian influenza A virus(H5) RNA stored and extracted from PrimeStore™ Solution, Viral TransportMedia, RNA Storage Solution, or nuclease-free water. PrimeStore™Solution extraction efficiency was compared to three commerciallyavailable nucleic acid extraction kits. Furthermore, the ability of RNAcontained in PrimeStore™ Solution to resist nuclease degradation wasevaluated.

Results

PrimeStore™ Solution (version 2, but lacking ethanol) inactivatedmicrobial agents while preserving released RNA/DNA from clinicalmaterial, i.e., nasal washes, throat swabs, or environmental samples.Clinical specimens or environmental samples placed in this solution werestabilized at room temperature for up to 30 days while degradation ofnucleic acids occurred in other transport media. PrimeStore™ Solution iscompatible with commercially available RNA isolation kits and producedan increased nucleic acid yield.

Example 8—Killing of MRSA (ATCC33592) in PrimeStore™ Solution

This example illustrates the effectiveness of the PrimeStore™ Solution(ver. 2.2) in killing a potential bacterial contaminant.Methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC33592 wasdiluted 10-fold and 1000-fold into PrimeStore™ Solution (Ver. 2.2) andquantitated (see FIG. 6).

Experimental Protocol

DAY PROCEDURE 0 Transfer MRSA (ATCC33592) from a Culti-loop ® (Remel) to1.5 mL of TSB in a 15-ml conical test tube. Incubate at 37° C. forapproximately 15 min. Gently vortex suspension and transfer 100 μL to ablood-agar plate. Incubate the plate overnight at 37° C. 1 Observe heavyand uniform colony growth after 12 hr incubation. Transfer ~10% ofcolonies to 300 mL of tryptic soy broth (TSB) in a sterile, 1-literflask. Place flask on shaker at 37° C. and 200 rpm. After approximately4-6 hrs' incubation, transfer ~50 mL of bacterial suspension to new1-liter flask containing 300 mL fresh TSB. After approximately 4-6 hrs'incubation, transfer ~100 μL of culture into 900 μL of TSB (1:10 controldilution). From this suspension 10 μL were transferred to 990 μL of TSB(1:1000 control dilution). Transfer 100 μL of culture into 900 μL ofPrimeStore ™ Solution (1:10 PrimeStore ™ dilution). From this suspension10 μL were transferred to 990 μL of TSB (1:1000 PrimeStore ™ dilution).Immediately after transfer into TSB or PrimeStore ™ Solution (ver. 2.2),the suspensions were gently vortexed and 100 μL were plated from bothdilutions of control and PrimeStore ™ suspensions onto blood agarplates. The time-zero time point was actually about two minutesfollowing addition of the bacteria to the TSB or PrimeStore ™ Solution.The suspensions in TSB and PrimeStore ™ Solution (ver. 2.2) were kept atroom temperature. An additional 100 μL was plated onto blood agar platesat 5, 15, 30, 60, 120 and 240 minutes after the preparation of thesuspensions in TSB and PrimeStore ™ Solution. Bacterial suspensions onthe plates were allowed to dry, the plated inverted and the platesincubated overnight at 37° C. A titration of the shaker culture was alsoperformed by mixing 100 μL of the suspension from the shaker culturewith 900 μL of TSB (10⁻¹ dilution). Serial 10-fold dilutions wereprepared through 10⁻⁹. 100 μL samples were plated onto blood agar fromall dilutions except 10⁻¹. 2 Plates were observed for bacterialcolonies. All plates were stored at 4° C. for later observation, ifnecessary.Results

The results are presented in Table 7 and Table 8. Briefly, the bacterialsuspension contained approximately 4.7×10⁹ cfu/ml. Thus, the 1:10dilution contained approximately 4.7×10⁸ cfu/ml and the 1:1000 dilutioncontained 4.7×10⁶ cfu/ml. At all time points, the bacteria suspended inTSB were too numerous to count. At all time points the bacteriasuspended in PrimeStore™ Solution and plated onto blood agar plates hadno detectable colonies.

TABLE 7 KILLING OF MRSA (ATCC 33592) BY PRIMESTORE ™ SOLUTION (VER. 2.2)Incubation Time In TSB In Primestore (Minutes) 1:10 1:1000 1:10 1:1000 0TNTC TNTC 0 0 5 TNTC TNTC 0 0 15 TNTC TNTC 0 0 30 TNTC TNTC 0 0 60 TNTCTNTC 0 0 120 TNTC TNTC 0 0 240 TNTC TNTC 0 0 TNTC = too numerous tocount.

TABLE 8 TITRATION OF MRSA ATCC33592 FROM SUSPENSION CULTURE DilutionCFU/plate CFU/ml 1.E+01 TNTC 1.E+02 TNTC 1.E+03 TNTC 1.E+04 TNTC 1.E+05TNTC 1.E+06 TNTC 1.E+07 35  3.5 × 10{circumflex over ( )}9 1.E+08 6   6× 10{circumflex over ( )}9 1.E+09 0 NOTE: CFU/ml calculations arecorrected to include the plating volume of 0.1 mls Final Conc: 4.7 ×10{circumflex over ( )}9/ml TNTC = too numerous to count. CFU = colonyforming units.An additional study was performed to determine the time of exposurenecessary for killing MRSA ATCC33592 when diluted 10-fold intoPrimeStore™ Solution (Ver. 2.2), and to determine the effect of dilutionof the bacteria after exposure to PrimeStore™ Solution, but beforeplating.Experimental ProtocolDay Procedure0 Transfer MRSA (ATCC33592) from TNTC plate from the study describedabove into 4 mL of TSB. These plates had been stored at 4° C. forapproximately 48 hrs. Bacteria were vortexed gently and placed at roomtemperature for approximately 10 min. before use.0.1 mL of bacterial suspension was transferred to 0.9 mL PrimeStore™Solution and vortexed gently. After approximately 60 sec, the bacteriain PrimeStore™ were again vortexed gently and0.1 mL of bacterial suspension was transferred into 0.3 mL of TSB (1:4dilution).100 μL of bacteria in PrimeStore™ Solution (designated “neat”) and fromthe 1:4 dilution into TSB were plated onto blood agar plates (5% sheepRBCs in TSA).This process was repeated at 5 and 15 min., and then again withdilutions made into TSB instead of PrimeStore™ Solution.The liquid bacterial suspensions on the blood agar plates were allowedto dry at room temperature and then incubated overnight at 37° C.1 After approximately 16 hrs. incubation, the plates were removed fromthe incubator and colonies counted.Results

The bacterial suspension contained an unknown number of colony formingunits (cfu) per mL. At all time points the bacteria suspended in trypticsoy broth (TSB) were too numerous to count (TNTC). At all timepoints thebacteria suspended in PrimeStore™ compositions and plated onto bloodagar plates produced no colonies (Table 9).

TABLE 9 KILLING OF MRSA (ATCC33592) BY PRIMESTORE ™ SOLUTION IncubationTime In TSB In TSB (minutes) neat 1:4 neat 1:4 1 TNTC TNTC 0 0 5 TNTCTNTC 0 0 15 TNTC TNTC 0 0 TNTC = too numerous to count.

Example 9—Additional Studies Evaluating PrimeStore™ Solutions

The data in FIG. 7B illustrate the ability of PSS to inactivatemicrobes. Shown is a study in which chicken cloacal specimens werecollected in PrimeStore™ Solution (Ver. 1). PrimeStore™ Solutioninactivated the microbial agents in 1 hr. Four original chicken cloacalsamples were immersed in PrimeStore™ Solution or water and subsequentlyplated on blood agar plates. These results showed that the disclosedcomposition could quickly kill or inactive microorganisms in the sample.

The data in FIG. 7C illustrate the ability of PSS to inhibit RNA basehydrolysis for 30 days at room temperature. RNA was incubated at roomtemperature (22-26° C.) in PrimeStore™ (gel lane 1 and 3) and water (gellane 2 and 4), and subsequently RT-PCR amplified (1500 base pairs) atDay 0 and Day 30. PrimeStore™ preserved collected RNA, and preventedRNA/DNA degradation at room temperature up to 30 days (see also, e.g.,Table 11).

Flu Inhibition Assay

The reagents for this assay include

Trypsin Medium:

45 mL Sterile N/C EMEM

3 mL stock 7.5% Na Bicarbonate (2%)

1.5 mL SPG (1%)

75 μL Trypsin (0.05%)

1.5 mL Fungizone (1%)

150 μL Gentamicin

Filter medium.

Crystal Violet:

150 ml glutaric dialdehyde

2 gm crystal violet

2850 mL deionized waterProtocols

Preparation of Serum Samples for Assay

Thaw and vortex serum samples. For each sample, label the lid of acorresponding Spin-X tube. Combine 450 μL non-complete EMEM with 50 μLserum into a Spin-X tube. Warm tubes containing the sera and EMEM in a56° C. water bath for 30 min. Centrifuge tubes at 8000 RPM for 2 min. atroom temperature. Label and place samples into a −20° C. freezer untilassayed.

Dilution Plates

Load 160 μL of each neat compound or serum sample into wells A1 throughA12. Load the remaining wells with 120 μL trypsin medium. Using amulti-channel pipette, draw 40 μL of neat sample from row A and diluteinto the corresponding wells in row B. Repeat dilution for each row,mixing well after each transfer. At row H, after mixing the transferfrom row G, draw up 40 μL from each well and discard. Obtain virus stock(10⁶) from −80° C. freezer and thaw. Dilute virus stock in trypsin mediato a 10³ dilution. After serial dilutions are completed, transfer 120 μLof influenza virus (10⁴ TCID₅₀ ml) to all wells in the dilution plate.This results in a total of 240 μL in all wells. Incubate dilutionplate(s) at room temperature for 1 hour.

MDCK Cell Plates

Sterilize and place glass reservoir, comb dispenser and tubing insidethe fume hood. Inside the hood, connect the tubing to the reservoir andfill nozzle of the comb. Connect the aspirator tube to the vacuum nozzleon the comb. Place the reservoir on an elevated surface and turn on theaspirator. Put PBS into the reservoir (1 L or more may be neededdepending on the number of plates). Wash the cell plates 3× with the PBScomb (aspirate the medium, then press the button for roughly 1 second towash the wells, repeat twice). Using a multi-channel pipette, transfer50 μL from each well in column 1 of the dilution plate to columns 1through 4 of the cell plate. Transfer 50 μL from each well in column 2of the dilution plate to columns 5 through 8 of the cell plate. Transfer50 μL from each well in column 3 to columns 9 through 12 of the cellplate. Repeat transfer to additional cell plates for remaining samples.Incubate cell plates for 1 hour at 37° C. After incubation period, add50 μL trypsin medium to all wells of the cell plates. Return plates toincubation chamber, and incubate for 4 days post-infection.

Staining

Add 100 μL of Crystal Violet to all wells. Let sit for 1 hour. Rinseplates in cold running water and air dry.

TABLE 10 TITRATION OF TCEP USING WHOLE INFLUENZA A VIRUS 5 mM 10 mM 25mM 35 mM 50 mM 30.353 24.58 24.52 24.14 25.9582 30.2261 22.74 24.2622.74 26.0337 30.28955 23.66 24.39 23.44 25.99595 0.089732 1.3010760.183848 0.989949 0.053387 Titration of TCEP Using H5 Avian ssRNA 27.225.25 25.63 27.3 28.3039 26.73 24.89 25.36 27.62 26.6854 26.965 25.0725.495 27.46 27.49465 0.33234 0.254558 0.190919 0.226274 1.144452Time-Course Study of the Long-Term Stability of PrimeStore Compositions

The following data demonstrate the effectiveness of various PrimeStorecompositions in preserving nucleic acid integrity over a thirty-dayperiod with samples stored at room temperature. PrimeStore compositionshave been compared to water alone, ethanol alone, commercial bufferssuch as VTM and AVL.

TABLE 11 30-DAY TIME-COURSE COMPARISON STUDY OF VARIOUS COMPOSITIONSPS-V1 PS-V1 PS-V2.2 VTM Water EtOH AVL (Year old) (new lot) PS-V2(w/EtOH) DAY 1 27.0225 26.1403 18.4463 24.2698 24.2607 23.9524 23.442620.2102 24.42 25.6044 18.3206 24.4789 24.3716 23.9615 23.7387 20.06325.72125 25.87235 18.4463 24.37435 24.31615 23.95695 23.59065 20.1366AVG 1.840245 0.378939 0.088883 0.147856 0.0784181 0.006435 0.2093740.10408612 STDEV DAY 6 29.1988 29.3053 27.4058 37.9226 27.2379 27.16524.53 22.4887 28.6799 28.7916 27.0781 40 26.4857 26.7658 24.4418 22.467628.93935 29.04845 27.24195 38.9613 26.8618 26.9654 24.4859 22.47815 AVG0.366918 0.363241 0.231719 1.468944 0.5318857 0.282277 0.0623670.01491995 STDEV DAY 12 27.997 28.151 26.9011 40 30.8352 31.0478 25.892622.2074 28.0062 28.2211 26.2139 38.0439 30.4502 30.1935 25.3037 22.002528.0016 28.18605 26.5575 39.02195 30.6427 30.62065 25.59815 22.10495 AVG0.006505 0.049568 0.485924 1.383172 0.2722361 0.604081 0.4164150.14488618 STDEV DAY 20 27.9851 28.7713 27.1105 40 30.1844 27.19325.7407 20.8364 28.4067 27.7929 27.0105 40 30.2465 27.2274 25.621320.2843 28.1959 28.2821 27.0605 40 30.21545 27.2102 25.681 20.56035 AVG0.298116 0.691833 0.070711 0 0.0439113 0.024324 0.084429 0.39039365STDEV DAY 30 29.23 31.9168 33.012 40 29.1993 30.2386 23.0589 20.934829.9067 31.3252 32.3001 40 28.827 29.6081 22.9662 20.4973 29.5683531.621 32.65605 40 29.01315 29.92335 23.01255 20.71605 AVG 0.4784990.418324 0.503389 0 0.2632559 0.445831 0.065549 0.30935922 STDEV PS-V1(year old) = One-year old PrimeStore Formulation (Ver. 1). PS-V1 (newlot) = Fresh PrimeStore Formulation (Ver. 2). PS-V2 = Fresh PrimeStoreFormulation (Ver 2) (without ethanol). PS-V2.2 (w/EtOH) = FreshPrimeStore Formulation (Ver. 2.2) (i.e. with ethanol).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of exemplary embodiments, it will be apparent tothose of ordinary skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically- and physiologically-related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those of ordinary skill in the art are deemedto be within the spirit, scope and concept of the invention as definedby the appended claims. Accordingly, the exclusive rights sought to bepatented are as described in the claims below.

f refrigeration or freezing prior to molecular analysis.

The invention claimed is:
 1. An aqueous composition that maintainsfidelity of a nucleic acid sequence contained with a biological samplewhen mixed with the sample and maintained for a period of time withoutrefrigeration, comprising components that upon mixing sterilize thesample, and denature proteins and inactivate nucleases of the sample,wherein the composition comprises guanidine thiocyanate,tris(2-carboxyethyl) phosphine (TCEP), sodium citrate, N-lauroylsarcosine, a silicone polymer, tris(hydroxymethyl) aminomethane (Tris),ethylenediaminetetraacetic (EDTA), and nuclease-free water in amountssuch that the fidelity of the nucleic acid sequence as determined by PCRis within three CTs within the period of time, and the period of time isat least 7 days.
 2. The composition of claim 1, wherein the sample isselected from the group consisting of blood, plasma, serum, sputum,urine, stool, buccal swabs, throat swabs, vaginal swabs, urethral swabs,cervical swabs, rectal swabs, lesion swabs, abscess swabs,nasopharyngeal swabs, tears, mucus, saliva, semen, vaginal fluids,lymphatic fluid, amniotic fluid, spinal fluid, cerebrospinal fluid,peritoneal effusions, pleural effusions, exudates, punctates, epithelialsmears, biopsies, bone marrow samples, fluid from a cyst or abscess,synovial fluid, vitreous or aqueous humor, eye washes or aspirates,pulmonary lavage or lung aspirates, an organ or tissue, and anycombination thereof.
 3. The composition of claim 1, wherein thecomposition comprises the guanidine thiocyanate in an amount from about0.5 M to about 6M, the TCEP is in an amount from about 0.5 mM to about30 mM, the N-lauroyl sarcosine in an amount from about 0.15% to about 1%(wt./vol.), the sodium citrate in an amount from about 0.5 mM to about50 mM, and the EDTA in an amount from about 0.1 mM to about 1.0 mM, andthe Tris comprises from about 1 mM to about 1 M.
 4. The composition ofclaim 3, wherein the composition further comprises ethanol.
 5. Thecomposition of claim 4, wherein the composition comprises the siliconepolymer in an amount from about 0.0001% to about 0.3% (wt./vol.), andthe ethanol in an amount from about 1% to about 25% (vol./vol.).
 6. Thecomposition of claim 1, wherein the period of time is at least 14 days.7. The composition of claim 1, wherein the period of time is at least 21days.
 8. The composition of claim 1, wherein the period of time is atleast 28 days.
 9. The composition of claim 1, wherein the period of timeis several months.
 10. The composition of claim 1, which is transportedfor analyzing fidelity of nucleic acids.
 11. The composition of claim 1,wherein the fidelity of the nucleic acid sequence is within two CTs. 12.The composition of claim 1, wherein the fidelity of the sequence iswithin three CTs for an RNA sequences and/or within two CTs for a DNAsequence.
 13. The mixture comprising the composition of claim 1 and abiological sample.
 14. The mixture of claim 13, which is safe forhandling and transportation.
 15. The mixture of claim 13, which ismaintained at room temperature.
 16. The mixture of claim 13, whereinroom temperature is from 15° C. to 30° C.
 17. The mixture of claim 13,wherein room temperature is from 18° C. to 28° C.
 18. An aqueouscomposition that maintains fidelity of a nucleic acid sequence containedwith a biological sample when mixed with the sample and maintained for aperiod of time without refrigeration, comprising components that uponmixing sterilize the sample, and denature proteins and inactivatesnucleases of the sample, wherein the composition comprises guanidinethiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate,N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic (EDTA), andnuclease-free water in amounts such that the fidelity of the nucleicacid sequence as determined by PCR is within three CTs within the periodof time for an RNA sequence and within two CTs for a DNA sequence withinthe period of timeand the period of time is at least 7 days.
 19. Thecomposition of claim 18, wherein the period of time is at least 14 days.20. The composition of claim 18, wherein the period of time is at least21 days.
 21. The composition of claim 18, wherein the period of time isat least 28 days.
 22. The composition of claim 18, wherein the period oftime is several months.
 23. The composition of claim 18, wherein the pHof the composition is from about 6 to about
 7. 24. The composition ofclaim 18, wherein the composition comprises the guanidine thiocyanate inan amount from about 0.5 M to about 6M, the TCEP is in an amount fromabout 0.5 mM to about 30 mM, the N-lauroyl sarcosine in an amount fromabout 0.1% to about 1% (wt./vol.), the sodium citrate in an amount fromabout 0.5 mM to about 0.3 M, and the EDTA in an amount from about 0.5 mMto about 50 mM.
 25. An aqueous composition comprising guanidinethiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate,N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic (EDTA), andnuclease-free water, wherein the pH of the composition is from about 6to about
 7. 26. The composition of claim 25, wherein the compositioncomprises the guanidine thiocyanate in an amount from about 0.5 M toabout 6M, the TCEP is in an amount from about 0.5 mM to about 30 mM, theN-lauroyl sarcosine in an amount from about 0.1% to about 1% (wt./vol.),the sodium citrate in an amount from about 0.5 mM to about 0.3 M, andthe EDTA in an amount from about 0.5 mM to about 50 mM.